1. Field of the Invention
This invention relates to a method for discriminating a kind of nucleic acid bases of DNA.
2. Related Background Art
As shown in FIGS. 1A-1D, DNA (deoxyribonucleic acid) is a copolymer of four kinds of deoxynucleotides, which are shown in FIGS. 2A-2D, i.e., deoxyadenylic acid (shown in FIG. 1A), deoxyguanylic acid (shown in FIG. 1B), deoxythymidylic acid (shown in FIG. 1C), and deoxycytidylic acid (shown in FIG. 1D). These deoxynucleotides have at the base sites of their respective deoxyriboses an adenine, a guanine, a thymine, or a cytosine, which are nucleic acid bases. A sequence of these deoxynucleotides is intrinsic to a gene.
Conventionally in sequencing these deoxynucleotides, a DNA chain is labeled using a radioactive isotope (R.I.) or a fluorescent dye. The conventional method is disclosed in U.S. Pat. No. 4,962,037. But this method needs a preparatory treatment of separately adding a fluorescent dye, etc.
In view of this, discriminating methods using the chromophores of DNA are used. As a typical example of these methods, a method using a solution of DNA in a water/methanol solvent with a ratio of 1:1 is described in "Basic Principles in Nucleic Acid Chemistry", Vol. I, Academic Press (1974), p. 322.about.328. In this method, a temperature of this solution is lowered to 77K, and then UV laser beams are irradiated to the sample solution by a secondary higher harmonic generator. By these laser beams directed to electrons of the respective chromophores of A (adenine), T (thymine), G (guanine), C (cytosine), four kinds of nucleic acid bases of each of DNAs are discriminated from each molecule flowing one after another in the above-mentioned solution. The reference discloses that the irradiation of laser beams as excitation light at a temperature below room temperature much improves quantum yields of the nucleic acid bases in comparison with yields at room temperature.
The solvent comprises a the mixture of water and methanol because, when water is used alone, cubical expansions of the water take place at low temperatures, breaking the container. Usually alcohol is added to water for the prevention of volume expansion at low temperatures, and, additionally, causes the solvent to be transparent and vitreous.
FIGS. 3A-3D show spectra of the fluorescence and phosphorescence emitted from the solution using the above-described method in A, T, G and C, respectively. As shown, all the nucleic acid bases have peaks of fluorescence near a wavelength of 325 nm. A and G have spectra having high peaks near 400 nm. These are phosphorescence spectra, and the lifetimes of the respective phosphorescence are of A and G 2.7 seconds, and 1.6 seconds. By their respective phosphorescence lifetimes, A and G can be discriminated from each other. T and C, which emit no phosphorescence, are measured in terms of lifetimes of fluorescence. FIG. 10 shows the lifetimes of fluorescence of the four kinds of nucleic acid bases. In FIGS. 4A-4D, fluorescence intensity is taken on the vertical axis, and lifetime of fluorescence is taken on the horizontal axis. 1 ch on the horizontal axis is equal to 77 psce. The sharp peaks around 100 ch show excitation light, and the blunt extinction curves of FIGS. 4A-4D respectively show the fluorescence from the nucleic acid bases A, T, G and C. In comparison with the fluorescence lifetime of T and that of C, which cannot be discriminated by phosphorescence, it is apparent that there is a difference in the extinction curve therebetween. Accordingly T and C can be discriminated from each other by fluorescence lifetime.
This method can be used to discriminate A from G by phosphorescence lifetime. However, but taking into account ultra-high speed sequencing, (at a speed at which one base can be identified per 1 second at worst), the respective phosphorescence lifetimes of A and G are too long to be used as a parameter for the discrimination of the nucleic acid bases.
The method discrimination by phosphorescence lifetime cannot be used for T or C, because T and C emit no phosphorescence. Accordingly T and C have to be discriminated from each other using respective fluorescence lifetimes. As a result, a problem exists in the current art that the nucleic acid bases contained in DNA cannot be discriminated from one another efficiently at ultra-high speed.
FIG. 5 shows relationships between fluorescence lifetimes of long lifetime components and ratios of the components. But as shown in FIG. 5, there are cases in which the discrimination is difficult, and made possible only by comparison of fluorescence lifetimes. Based on the long lifetime components, the ratio of the fluorescence lifetime 365 ps of C is 77.6%, which can be apparently discriminated from the other three nucleic acids A, T, G. But it is difficult to discriminate A, T, G by long lifetime components. Furthermore, even if G can be discriminated, it will be difficult to discriminate A from T.